A gene for Hirschsprung disease maps to the proximal long arm of chromosome 10.

Hirschsprung disease (HSCR) is a frequent congenital disorder (1 in 5,000 newborns) of unknown origin characterized by the absence of parasympathetic intrinsic ganglion cells of the hindgut. Taking advantage of a proximal deletion of chromosome 10q (del 10q11.2-q21.2) in a patient with total colonic aganglionosis, and of a high-density genetic map of microsatellite DNA markers, we performed genetic linkage analysis in 15 non-syndromic long-segment and short-segment HSCR families. Multipoint linkage analysis indicated that the most likely location for a HSCR locus is between loci D10S208 and D10S196, suggesting that a dominant gene for HSCR maps to 10q11.2, a region to which other neural crest defects have been mapped.

Search for pathogenetic variants of the SPRY2 gene in intestinal innervation defects.

SPRY2 is an inducible inhibitor of signalling mediated by tyrosine kinases receptors, whose targeting causes intestinal hyperganglionosis in mice. In this light, we have undertaken a mutational analysis of the SPRY2 gene in patients affected with intestinal neuronal dysplasia (IND), without detecting nucleotide changes in any of the 26 DNA samples analysed, with the exception of two already known polymorphic variants. A role of the SPRY2 gene in IND pathogenesis can be thus excluded.

Conservative management of chylous ascites after oncological surgery for peripheral neuroblastic tumors in pediatric patients.

Chylous ascites may complicate the postoperative course of abdominal surgery mainly due to the iatrogenic disruption of the lymphatic channels during extensive retroperitoneal dissection. Sparse data are available regarding treatment; however, in many cases a recommended first-line treatment approach is by way of enteral feeding, consisting of a formula high in medium-chain triglycerides (MCTs) together with a complete total parenteral nutrition teamed with somatostatin (or an equivalent). Nonetheless, the ligation of chylous fistulae, together with the application of Fibrin glue, as well as the creation of peritoneal-venous shunts have also been documented. The aims of this study are to document incidence of postoperative chylous ascites following resection of abdominal peripheral neuroblastic tumors, evaluate efficacy of the management of chylous ascites, and investigate the main risk factors. A survey was carried out over a span of six years, from March 2010 to March 2016 at Giannina Gaslini Children's Hospital involving seventy-seven children with resections of peripheral neuroblastic tumors. Incidence rate of postoperative chylous ascites following a normal diet was 9% (n=7). Treatment using total parenteral nutrition with octreotide resulted in a complete recovery from chylous ascites within a 20 day period without recurrence. Length of operative time, nephrectomy, and the extension of lymphadenectomy were all significantly associated with a higher incidence of postoperative chylous ascites (p<0.05) which also lengthened hospital stay (p<0.05) and possibly delayed beginning adjuvant chemotherapy.

Hirschsprung's disease, one of the most difficult diagnoses in pediatric surgery: a review of the problems from clinical practice to the bench.

PURPOSE: The diagnosis of Hirschsprung's disease (HSCR) should take place early in the neonatal period, because without an effective diagnosis and appropriate treatment, a considerable proportion of infants will go on to develop serious complications such as acute enterocolitis or toxic megacolon. Because no more than 10 % of HSCR cases have a late presentation with classical chronic constipation and megacolon, the clinician has to make a difficult, early diagnosis, which is the crux of the clinical problem. The aim of this review paper is to present all tools currently available to make a clear HSCR diagnosis and to discuss the problems facing the clinician and the pediatric surgeon in the correct identification of HSCR and of other intestinal dysganglionoses. METHODS: Based on the current state of knowledge and 24 years' personal experience in clinical practice and basic research in this field, I describe an algorithmic approach that enables clinicians and surgeons to rationalize and maximize the clarity of diagnosis through a complementary set of procedures and enzyme-histochemical reactions. RESULTS: Two innovative techniques, added to the protocol in the last four years, are described: the lyophilized HSCR diagnostic kit, and the one-trocar transumbilical laparoscopic intestinal full-thickness biopsy technique (OTTLB). CONCLUSION: The rational, algorithmic diagnostic pathway proposed in this review paper aims to optimize every diagnosis by the stepwise application of a complementary set of procedures and enzyme-histochemical reactions as they become appropriate. In the interests of simplifying genetic molecular diagnosis, I suggest the following guidelines: 1) only in cases of total colonic aganglionosis (TCA) is it advisable to carry out full RET mutation screening (the mutation rate is up to 70 %); and 2) all HSCR patients should be tested only for standard MEN2A and MTC mutations. If these are present, the patients should be followed up carefully with proper surveillance and biochemical testing of other susceptible family members as they are at risk of developing neuroendocrine tumors.

Neural crest neuroblasts can colonise aganglionic and ganglionic gut in vivo.

INTRODUCTION: Neural crest (NC) cells differentiate IN VITRO into neuroblasts, precursors of the enteric nervous system (ENS), when stimulated by specific agents. We developed a study aimed at establishing whether NC-derived neuroblasts can survive and colonise IN VIVO when injected into a recipient mouse gut. MATERIALS AND METHODS: The neuroblast precursors of the ENS were obtained from the vagal portion of the neural tubes of 296 CD-1 and GTROSA26 mouse embryos. The embryonic cells of GTROSA26 mice are identifiable through beta-galactosidase activity which allows recognition by blue staining. The host used in this study was the DOM/+ mouse, an animal model for Hirschsprung's disease (aganglionic megacolon). DOM/+ mouse pups (n = 43) received NC-derived cells inoculated into the seromuscular layer of the gut (33/43) or directly into the peritoneal abdominal cavity (10/43). RESULTS: All DOM/+ mice survived the procedure and were sacrificed after 7 or 14 days. Histochemical staining detected implanted cells in all mice. These showed specific myenteric colonisation into the aganglionic and ganglionic gut. CONCLUSION: The striking result of this study was the specific tropism of the injected NC-derived cells to target sites under the action of unknown chemotactic agents. This experimental procedure might represent a possible treatment option for specific forms of human ENS anomaly such as total intestinal aganglionosis.

Uncombable hair, retinal pigmentary dystrophy, dental anomalies, and brachydactyly: report of a new patient with additional findings.

A new ectodermal dysplasia syndrome was reported by Bork et al. in 1987 (Hautarzt 38:342-347). The syndrome consisted of hypotrichosis with the typical SEM (scanning electron microscopy) changes of uncombable hair, retinal pigmentary dystrophy, juvenile cataract, oligodontia, brachydactyly with brachymetacarpia; it was inherited as an autosomal dominant trait. We describe a sporadic case and add further clinical findings to expand the spectrum of this rare syndrome.

Congenital diaphragmatic hernia associated with ipsilateral upper limb reduction defects: report of a case with thumb hypoplasia.

Four unrelated cases of congenital diaphragmatic hernia associated with ipsilateral upper limb reduction defects were reported by McCredie and Reid in 1978 (J Pediatr 92: 762-765). As contiguous segments of the cervical neural crest are involved in the development of diaphragm and arms, the authors suggested that an early injury to the cervical neural crest might be the common underlying pathogenesis. We describe here a further example of this malformation complex: a newborn with a left posterolateral diaphragmatic hernia associated with ipsilateral thumb hypoplasia.

Total colonic aganglionosis.

The authors describe the genetic, pathophysiology, diagnostic, and therapeutic aspects of total colonic aganglionosis and of aganglionosis extending to the small intestine. The pathogenesis of this disease is genetically determined and is related to the differentiation and migration of cells derived from neural crests. The clinical and radiological features can be useful in the diagnosis but they are not pathognomonic. The histochemical estimation of acetylcholinesterase activity in suction rectal biopsies is useful in establishing the diagnosis; however, the specimens should be examined by an experienced pathologist. The definitive diagnosis of either condition is obtained by performing intraoperative seromuscular biopsies of the rectum, colon, and ileum. From the therapeutic point of view, many surgical techniques have been proposed for the radical treatment of this disease. Some of the techniques have been derived from operations proposed for the treatment of classic Hirschsprung's disease; others have been specifically designed.

The genetics of anorectal malformations: a complex matter.

Because the spectrum of anorectal malformations is wide, genetic investigations of these anomalies should include the study of multigenic models presenting variable penetrance and expressivity. Current knowledge in clinical genetics, cytogenetics, and molecular genetics of anorectal anomalies are reviewed. The analysis of associated anomalies (that are found in more than 60% of anorectal malformations) is an important aspect of the molecular study, because the association of anomalies with mendelian transmission or with a recognized causative gene can be an essential starting point for further investigations. In the present study, the authors focus on associated sacral anomalies, urethral malformations, and intestinal dysganglionoses. In particular, associated sacral anomalies could be a partial expression of the Currarino syndrome, which represents the only association for which genetic evidence has been demonstrated by linkage analysis. The authors studied a four-generation pedigree with recurrence of the Currarino syndrome, and the haplotype reconstruction confirmed that the gene segregating in this family is located in the 7q36 region. The collection and study of families with multiple cases of anorectal malformations could show whether different phenotypes are caused by single genes.

Immunohistochemical localization of RET protein in Hirschsprung's disease.

A major gene causing Hirschsprung's disease was recently mapped in 10q11.2. Its physical localization was restricted to a 250-Kb interval containing the RET proto-oncogene (REarranged during Transfection). In 1994, point mutations affecting the RET proto-oncogene were identified in patients with Hirschsprung's disease. The authors present an immunohistochemical study on the expression and localization of the Ret protein (a receptor tyrosine kinase, which is the RET proto-oncogene product) in the intestinal plexuses of patients with Hirschsprung's disease. Ninety-two full-thickness intestinal wall pieces from 29 pediatric patients were studied (19 cases of classic Hirschsprung's disease, 5 of total colonic aganglionosis, and 5 controls). Ret protein immunohistochemical localization was obtained using c-Ret R5, anti-Ret K and anti-Ret C antibodies, respectively, against the extracellular domain, the tyrosine kinase domain, and the carboxy-terminal 20 amino acids of the Ret protein. A diffuse granular staining was present in the ganglia of normal colon, whereas the small ganglia of the hypoganglionic colon showed a reduced number of ganglion cells that were strongly stained with c-Ret R5 MoAb. A reduced synthesis of Ret protein was shown in the ganglionic and hypoganglionic segments of two cases of this series, the first with a complete deletion of the RET proto-oncogene and the second with a frameshift mutation and a stop codon in the extracellular domain. The activity of the receptor tyrosine kinases (RTKs) in intestinal ganglion cells was investigated using antiphosphotyrosine antibodies. A very low tyrosine kinase activity was shown in the small ganglia of the hypoganglionic segment.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathogenesis of Hirschsprung's disease.

Hirschsprung's disease is an inherited disorder showing incomplete penetrance and variable expressivity. Genetic mapping and mutation screening of candidate genes, together with the study of several natural and knockout animal models, clearly have shown the involvement of several different genes in the pathogenesis of Hirschsprung's disease. Among these genes, the RET proto-oncogene accounts for the highest proportion of both familial and sporadic cases, with a wide range of mutations scattered along its entire coding region. The low detection rate of RET mutations in Hirschsprung patients also led to different hypotheses, such as the existence of additional Hirschsprung genes. Different animal and human genetic studies have identified 6 Hirschsprung genes: RET proto-oncogene (RET), endothelin 3 (EDN3), endothelin B receptor gene (EDNRB), glial-cell-line-derived neurotrophic factor (GDNF), endothelin converting enzyme (ECE1), gene encoding the Sry-related transcription factor SOX10 (SOX10). Microenvironmental factors also can play a role in the pathogenesis of aganglionosis. The developmental process of the crest-derived progenitor cells is sensitive to the level of different molecules. The expression deficit of different factors (GDNF, NTN) in the hindgut, in the absence of genetic mutations, could determine a missed activation of the receptor system, causing enteric neuroblast migration arrest.

Solo-RBT: a new instrument for rectal suction biopsies in the diagnosis of Hirschsprung's disease.

BACKGROUND/PURPOSE: Preoperative histochemistry on rectal mucosal-submucosal specimens is the most important step in the diagnosis of Hirschsprung's disease and other dysganglionoses. Today, rectal mucosal-submucosal biopsy specimens are obtained by suction with the widely used tool first designed by Noblett in the late 1960s. The authors developed a new instrument, for which a patent has already been filed, for one-hand execution of rectal suction biopsies, which will make this operation easier and faster. METHODS: The authors modified the old tool but maintained the cutting method described by Noblett. The authors' innovative automatic tool (Solo-RBT) is based on the possibility of sequencing suction and cutting of the rectal mucosa just by pressing a "trigger." The trigger activates a plunger to obtain the vacuum necessary for the suction and a cutter for mucosal sampling. To perform a correct biopsy in children with different weight and in adults, Solo-RBT can be adjusted suitably to obtain automatically the vacuum required in each case. Additionally, this tool is provided with 2 different sizes of both capsules and cutters, and it can be disassembled completely for easy cleaning, maintenance, and sterilization. RESULTS: The instrument was tested clinically on 62 children (189 biopsies), and it proved to be versatile, atraumatic, and extremely handy, because it can be used by just 1 physician who only needs to press the trigger to obtain the biopsy. CONCLUSIONS: Solo-RBT not only maintains the benefits (atraumatic, easy to use, rapid) and the main characteristic (cutting system) of Noblett's tool but it also improves its features and eliminates most of its disadvantages. Therefore, Solo-RBT represents a significant improvement in the diagnosis of Hirschsprung's disease and other dysganglionoses.

Ret protein in the human fetal rectum.

A major gene for Hirschsprung's disease (HD) recently has been mapped in chromosome 10q11.2 and identified to be the RET proto-oncogene. Mutations of the RET gene have occurred in HD patients, and abnormalities of expression and function of Ret protein (a receptor tyrosine kinase, which is the product of the RET gene) have been found in their intestines. In vitro studies of the biological effects of HD mutations suggest a loss of function effect, which may be negative-dominant. However, the developmental role of the Ret protein in the organogenesis of the enteric nervous system (ENS) and its role in the pathogenesis of HD remain unclear. The authors present a study of the expression of Ret protein in the human ENS during fetal development. Fresh rectal tissues were obtained from nine fetuses (gestational age range, 12 to 22 weeks). Ret protein expression was studied immunohistochemically, using antibodies against the carboxy-terminal 20 amino acids (anti-Ret C) and the extracellular domain (anti-Ret R5). The tyrosine kinase activity of the fetal ENS was investigated with antiphosphotyrosine mouse monoclonal antibody against the phosphorylated tyrosine residues. Anti-Ret C immunostaining was observed in ganglion cells at all ages, but intense activity was significantly higher among the cells of the younger fetuses. Intense anti-Ret R5 immunostaining was present in the enteric ganglion cells of the 12-week-old fetus. The tyrosine kinase activity of ganglion cells increases progressively with advancing gestational age. The results of this study support the hypothesis that the Ret protein receptor might play a crucial role in the cellular and molecular processes involved in the development and maturation of the ENS, abnormalities of which could result in HD. High Ret protein expression and low tyrosine kinase activity have been reported to occur in small ganglia of the HD hypoganglionic segment. In the present study, these markers were typical of the primitive and immature ENS during the early phase of hindgut development.

Anorectal malformations associated with enteric dysganglionosis in Danforth's short tail (Sd) mice.

BACKGROUND: The spontaneous mutant Danforth's short tail (Sd) mouse has been studied over the last 60 years from the morphological, embryological, and genetic point of view. The Sd mutation affects a gene essential to notochordal development, and the Sd mouse phenotype represents an analogue of human caudal regression syndrome. The Sd/Sd mouse presents different types of anorectal malformations (ARM) and was suggested as a simple and cheap model of investigation of ARM morphology and embryology. In the current study, the Sd mouse enteric nervous system (ENS) was thoroughly investigated with specific immunohistochemical markers. METHODS: Macroscopic analysis, normal histology, and immunohistochemical techniques for detecting neurofilaments (NF) and NOS1 were used to study ENS of 138 Sd mice and 25 controls. RESULTS: The surprising results of this study showed that Sd mutation is associated with different degrees of hypoganglionosis and aganglionosis. In 41% of Sd/SD-affected mice, the rectal pouch was aganglionic and in the remaining 58% was severely hypoganglionic. In addition, 4.1% of heterozygous mice presented a distal aganglionosis and 8.3% hypoganglionosis. CONCLUSIONS: These results suggest that Sd mutation independently affects distinct cell lines during early organogenesis, as notochord cells, ventral hingut endoderm, and neuroblasts migrating from neural crest cells. Comparing the Sd murine model with human pathology, this study confirms that the association between ARM and intestinal dysganglionosis is not rare and underlines the importance of detecting in every ARM patient the innervation abnormalities of rectal pouch and fistulas.

GDNF deficit in Hirschsprung's disease.

BACKGROUND/PURPOSE: In 1996, the glial cell line-derived neurotrophic factor (GDNF) was identified as one of the ligands of the RET transmembrane receptor. In the same year, GDNF mutations were found in association with RET protooncogene mutations in Hirschsprung patients. Mutations in GDNF per se are thought neither necessary nor sufficient to cause Hirschsprung's disease (HD). To date, our study group has identified GDNF mutations only in 2 of 98 cases of intestinal dysganglionosis. The aim of our study was to investigate a possible expression deficit of GDNF in the enteric nervous system of Hirschsprung patients not mutated for the GDNF gene. METHODS: We used rabbit polyclonal antibodies raised against a peptide corresponding to amino acids 186-205 mapping within the carboxy-terminal domain of human GDNF. GDNF expression was studied immunohistochemically in surgical specimens from 30 HD cases (27 classic forms and 3 ultralong forms) and from 10 age-matched controls. Serial sections from the same full-thickness specimens were investigated with the following histochemical and immunohistochemical techniques: acetylcholinesterase, lactate dehydrogenase, succinic dehydrogenase, alpha-naphthyl-esterase, glial fibrillary acid protein, S-100 protein, and neuron-specific enolase. RESULTS: A high level of GDNF expression was found in normal intestine and in Hirschsprung ganglionic segment. Satellite elements of myenteric ganglia presented a strong immunoreactivity to GDNF. Conversely, the aganglionic segment showed cholinergic hyperinnervation and hypertrophic trunks of nerve fibers in the muscular interstitium with complete absence of GDNF expression. The small ganglia of the hypoganglionic segment showed a reduced GDNF immunoreactivity. CONCLUSIONS: GDNF, a distantly related member of the transforming growth factor-beta superfamily, is a potent neurotrophic and survival factor for neurons and enteric ganglion cells. Mutations of the GDNF gene or GDNF expression deficit interrupt the faithful GDNF signaling via Ret, contributing to HD pathogenesis.

Use of staplers in congenital lobar emphysema.

The authors report on a technique for the treatment of congenital lobar emphysema in a patient of 6.5 kg body weight. After left thoracotomy and vessel isolation, a section of the lobar bronchus and interlobar fissure was performed, because of incompleteness of 3/4 of its length. Metal clips were used with the stapler ENDO-GIA 30v Autosuture after tissue calibration. The authors underline the importance of the staplers, which have been widely used in adult surgery, because they reduce the risk of bacterial contamination and air leakage through the suture rims, especially in lung resective surgery.

A new rapid acetylcholinesterase histochemical method for the intraoperative diagnosis of Hirschsprung's disease and intestinal neuronal dysplasia.

The most commonly used acetylcholinesterase (AChE) method for the diagnosis of Hirschsprung's disease (HD) and intestinal neuronal dysplasia (IND) was first introduced in 1964 by Morris Karnovsky and Logan Roots. This technique requires about 80 - 120 minutes incubation time and cannot be used for the intraoperative diagnosis of HD and IND. To avoid these limitations, in 1994 Kobayashi et al first proposed an accelerated modified method in two different versions, the first using diaminobenzydine (DAB) reagent, the second using 4-chloro-1-naphthol as final reagent. In the present study, we propose a new rapid variation of AChE staining which avoids the use of DAB and naphthol, notably toxic reagents, but follows the same acceleration principle of Kobayashi's technique. Our modified rapid AChE requires a total incubation time of only 8 minutes, which is compatible with intraoperative histochemical examination purposes. Intraoperative seromuscular or full-thickness intestinal biopsies were obtained from 92 children affected by intestinal dysganglionoses. The biopsies were frozen and cut in 15 microm cryostatic sections. Rapid AChE was performed with a special incubation medium using 3-amino-9 ethylcarbazole (AEC) as chromogenic substance. The two complementary histochemical techniques alpha-naphthylesterase (ANE) and lactate-dehydrogenase (LDH) were also used intraoperatively for the staining of ganglion cells. The diagnosis was confirmed postoperatively with conventional AChE Karnovsky technique, comparing the extensions of hyperganglionic, hypoganglionic and aganglionic segments in each studied case. The new rapid AChE modified method can identify ganglion cells and fibers using a dark brown precipitate. In all the cases studied, the intestinal innervation pattern identified with this modified technique was similar to that obtained with Karnovsky AchE. Seventy-eight HD, 8 isolated IND and 6 HD associated with an evident IND segment were diagnosed. This new rapid AChE histochemical technique avoids the use of DAB and naphthol, and can thus be considered safe for operators. Rapid AChE is a valid tool for both the evaluation of aganglionosis extension and for the identification of IND pattern during surgery. We recommend this very reliable method for the intraoperative diagnosis of HD and IND, in association with other enzymatic markers of ganglion cells (ANE or LDH). We propose the following diagnostic protocols: a) for preoperative histochemical study: conventional AChE plus LDH and NADPH-diaphorase; b) for intraoperative study: rapid AChE plus ANE.

Long-gap oesophageal atresia: a combined endoscopic and radiologic evaluation.

The final aim of the treatment of oesophageal atresia is to restore continuity without the interposition of intestinal segments. The authors present the results of a new diagnostic method combining endoscopy and radiology to evaluate the development of pouches in oesophageal atresia. This new method has been successfully adopted in four patients aged between 4 and 8 months, three with type I and one with type III complicated atresia. Direct end-to-end anastomosis was performed in all patients. We think this method can be easily applied and is a suitable diagnostic examination in all cases in which surgical strategy aims at restoring oesophageal continuity by means of direct anastomosis.

Neuronal intestinal dysplasia: clinical experience in Italian patients.

The authors present a review of 431 children biopsied and studied with the following histochemical and immunohistochemical techniques: 1) acetylcholinesterase activity; 2) alphanaphthylesterase activity; 3) S-100 protein immunohistochemical technique; 4) glyoxylic acid method. Two hundred forty-eight patients of our series presented different forms of dysganglionosis, 12 of them (4.8%) presenting neuronal intestinal dysplasia type B. In 7 cases, NID type B was diffuse, whereas in 5 recto-colonic NID type B was confined to the splenic flexure. Male:female ratio was 9:3. Familial recurrence was present in 2 of the 12 cases of our series, affected by severe neuronal intestinal dysplasia extended to the small intestine, associated with intestinal malrotation and short bowel syndrome. Four of the 7 cases of diffuse NID type B and 2 of the 5 cases of rectocolonic NID type B were surgically treated. Three patients with diffuse NID died from sepsis within the 2nd year of life. This study confirms that NID type B is a form of dysganglionosis which can be diagnosed in a Mediterranean country if histochemical techniques are applied in the study of a large series of constipated and pseudo-Hirschsprung patients. From a pathogenetic point of view, the authors compared the histochemical findings of biopsies from their series of NID patients with those of recto-colonic biopsies from patients with MEN II B syndrome. The similarity of GI symptoms in MEN II B and NID pediatric patients suggests that the two disorders could be the result of mutations affecting the same domain of the RET proto-oncogene.